An Energy Frontier Research Center

Transcription

1 An Energy Frontier Research Center Mission: to develop a nano-scale understanding of the structure and formation of lignocellulose, the main structural material in plants, forming a foundation for significant advances in sustainable energy and materials.

2 Theme 1: Cellulose synthesis & structure 1. Structure & function cellulose synthase (CESA) 2. Structure, formation and function of cellulose synthase complex (CSC) 3. Formation of microfibril from emerging glucans 4. Microfibril structure 5. Intercalation of matrix polysaccharides Theme 2: Structure & assembly of cell walls 6. Matrix-microfibril binding 7. Matrix-matrix interactions 8. Rearrangements during growth and maturation 9. Bundling of microfibrils -> macrofibrils & 2D networks 10. Physico-chemical basis for stiffening by lignin 11. Nano > meso scale structure & mechanics

3 Stoichiometry of the Plant Cellulose Synthesis Complex BT Nixon, K Mansouri, A Singh, J Du, JK Davis, JG Lee, E Slabaugh, VG Vandavasi, H O Neill, EM Roberts, AW Roberts, YG Yingling, CH Haigler Scientific Achievement We combined multidisciplinary approaches to show that a maximum of 18 CESAs can pack into the plant cellulose synthesis complex (CSC). This overturns long-standing dogma that CSCs contain 36 CESAs and microfibrils contain 36 glucans. Research Results New tools: superior CSC images; image averaging; CESA model with 7-TMH (computational) Spatial overlay of the negativelystained ratcesa1 catalytic trimer with SAXS (yellow) and in silico (blue) model cross-sections Significance Size matters. The results imply that cellulose is synthesized as an 18-chain fundamental fibril. An 18-chain fibril has high capacity for aggregate to form larger fibrils and networks and to interact with cell wall matrix components. A small fibril increases flexibility for assembly of diverse plant cell walls and likely impacts biomass properties for energy and material purposes. A hexamer of modeled trimers fit the average image well. Nixon et al Comparative Structural and Computational Analysis Supports Eighteen Cellulose Synthases in the Plant Cellulose Synthesis Complex. Scientific Reports 6, 28696; Related publication: Vandavasi et al Plant Physiology 170:

4 Plant Cellulose Synthases Form Cellulose Microfibrils in Vitro P Purushotham, SH Cho, S Moreno, V Bulone, M Kumar, T Nixon, J Zimmer Scientific Achievement Expression of poplar CesA8 and moss CesA5 in yeast yields active enzymes that form microfibrils. No other plant-specific factors are required for cellulose biosynthesis. Research Details - Purified, recombinant CesAs synthesize cellulose in vitro. - The cellulosic material is organized into microfibrils. - Removal of an N-terminal dimerization motif does not affect glucan synthesis but abolishes microfibril formation. - Suggests that CesA can form homo-oligomer in vitro, whereas hetero-trimeric complexes are likely formed in vivo. Purushotham et al A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro. PNAS 113, ; Cho et al., in review Significance First successful biosynthesis of cellulose from purified plant enzymes > sets the stage for mechanistic studies. A single plant CesA isomer can form microfibrils, which means that heterotrimeric CSCs & other proteins are not required for microfibril formation.

5 High-Field 2D Solid-State NMR Reveals Cellulose Structural Polymorphisms T Wang, H Yang, JD Kubicki, YB Park, DJ Cosgrove, M Hong Scientific Achievement Cellulose from primary cell walls was found to be more complex than that of highly crystalline celluloses (algae, plant 2 o walls, bacteria) and differs from allomorphs 1α and 1β. (a) Cellulose polymorphism: seven forms Results Primary cell walls were measured by 2D high-field SSNMR. DFT calculations were used to match data with molecular structures. Five types of interior cellulose (a-e) and two types of surface cellulose (f, g) mix in the same microfibril. Cellulose-d interacts with xyloglucan, is dehydrated, and is targeted by the wall loosening protein expansin. (b) Spatial proximities (c) Cross-sectional model Significance Knowing cellulose complexity is essential for models of cell wall structure and for investigating mechanisms of cell wall assembly. Wang et al Cellulose Structural Polymorphism in Plant Primary Cell Walls Investigated by High-Field 2D Solid-State NMR spectroscopy and Density Functional Theory Calculations. Biomacromolecules 17, ; & earlier publications.

6 Nanoscale movements of cellulose microfibrils in extending cell walls T Zhang, D Valvylonis, DM Duracho, B Tittmann, DJ Cosgrove, Scientific Achievement Discovered that motions of individual microfibrils differed for forceinduced and enzyme-induced cell wall strain, revealing for the first time - meso-scale connectivity between cellulose microfibrils. A Research Details A: Microfibrils of single-layer cell wall strips were imaged by AFM. B: Automated image analysis identified and quantified microfibrils motions. C: Simultaneous nanomechanical mapping (heat-map) revealed that tensile stresses were borne by both microfibrils and matrix. Significance - Shows that elastic strains are fundamentally different from the motions occurring during cell wall creep (ergo growth). - Reveals how microfibrils are connected to each other and to matrix. - Calls for a different modeling approach to incorporate microfibril motions and altered microfibril connectivities upon cell wall loosening. T Zhang et al Nanoscale movements of cellulose microfibrils in primary cell walls. Nature Plants 3, 17056; and previous publications B C

7 New tool/techniques: Combined AFM, extensometer & advanced image analysis Imaging of submerged walls by atomic force microscopy Extensometer (force/extension) built onto AFM stage FORCE POSITION newly-deposited cell wall surface Inner cell wall surface Cantilever 1. Advances in sample preparation and AFM technique improve microfibril imaging. 2. Construction of a device to stretch the cell wall on the AFM stage. 3. Well-defined treatments to produce different strains: plastic, elastic, stress relaxation, creep. START PLASTIC ELASTIC AFTER CREEP

8 CLSF Talks: 1. IN VITRO RECONSTITUTION OF PLANT CELLULOSE BIOSYNTHESIS (I-I-3; M 3:40) Presented by Jochen Zimmer: work from University of Virginia, Penn State University; and Royal Inst Technology 2. THE NANOMACHINE THAT SYNTHESIZES CELLULOSE IN PLANTS (A-II-1; T 8:30) TEAM SCIENCE COMPETITION Abhishek Singh, Jason N. Burris, Xiaoran Xin, Hui Yang et al: work from NC State U; U RI, Penn State U, UT El Paso, U VA 3. ALL-ATOM STRUCTURAL MODEL OF PLANT CELLULOSE SYNTHASE AND CELLULOSE SYNTHASE COMPLEX (A-II-5 T 9:50) Presented by Yaraslova.G. Yingling: work from NC State U; Penn State U; Oak Ridge NL; U RI; and U KY 4. CELLULOSE STRUCTURE, ORGANIZATION AND INTERACTIONS WITH MATRIX POLYSACCHARIDES & PROTEINS (A-II-3 T 9:10) Presented by Daniel J. Cosgrove: work from MIT, Penn State University; and University of Texas at El Paso

9 CLSF Posters: 1. CELLULOSE ASSEMBLY IN PLANT CELL WALLS IN THEIR NATIVE STATES NEW INSIGHTS AND NEW QUESTIONS FROM SUM- FREQUENCY-GENERATION VIBRATIONAL SPECTROSCOPY Seong H. Kim, S.Huang, M.Makarem, S. Kiemle, D.J. Cosgrove, C.Xiao, C.T. Anderson, X. Xin, Y. Gu; Penn State U. Mon 2. PROBING LIGNIN EFFECTS ON THE PLANT CELL WALL ULTRASTRUCTURE AND MECHANICAL PROPERTIES Sarah N. Kiemle, C.J. Liu, L.A. Wilson, D.J. Cosgrove ; Penn State U and Stony Brook U. 3. THE STRUCTURAL ROLE OF PECTINS IN CELL WALL LOOSENING FROM SOLID-STATE NMR SPECTROSCOPY Pyae Phyo, T. Wang, S. Kiemle, C. Xiao, C.T. Anderson, D.J. Cosgrove and Mei Hong; MIT and Penn State U. Tues 4. TRANS-DISCIPLINARY STRUCTURAL ANALYSIS PROVIDES INSIGHT INTO FUNCTION AND VARIATION OF THE PLANT CELLULOSE SYNTHESIS COMPLEX Candace H. Haigler, B.T. Nixon, J. Du, J.K. Davis, J.N. Burris, A. Singh, Y.G. Yingling, H. O'Neill, E.M. Roberts, A.M. Chaves, A.W. Roberts; NC State U; Penn State; Oak Ridge NL; RI College; U RI. 5. PRODUCTION AND ORGANIZATION OF CELLULOSE MICROFIBRILS IN PRIMARY AND SECONDARY CELL WALLS S. Li, X. Xin, Y. Zheng, T. Zhang, S. Huang, S. V. Pingali, S.H. Kim, H. O Neill, D.J. Cosgrove, and Ying Gu; Penn State U; Oak Ridge NL.

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